Supplemental Method information
Selection and preparation of lymphocyte samples
Due to pregnancy and study-related complications, cord blood samples of sufficient quantities for cell
isolation were collected from only 83 participants. Among these 83 participants, only 66 maternal T
lymphocyte DNA samples were of sufficient quantities for DNA methylation analyses. From these T
lymphocyte DNA samples, 44 cord blood samples of non-medicated women with singleton pregnancies
were selected for the genome-wide DNA methylation analyses together with 38 antepartum maternal
samples.
Whole blood was collected in heparin-coated tubes, stored on ice, and processed within a day of
collection. The peripheral blood mononuclear cell (PBMC) layer was separated by centrifugation at 400
g with Ficoll-Paque (GE Healthcare, Little Chalfont, UK) following previously published protocol. 25
After the washing the PBMCs with Hank’s Balanced Salt Solution (HBSS; Invitrogen, Carlsbad, CA,
USA), CD19+ B lymphocytes and CD3+ T lymphocytes were isolated from the PBMCs with
Dynabeads® CD19 pan B (111.43D, Invitrogen, Carlsbad, CA, USA) and Dynabeads® CD3 (111.51D,
Invitrogen, Carlsbad, CA, USA) respectively using a strong magnet (Stemcell Technology, Vancouver,
BC, Canada) and washed with 4% fetal bovine serum (FBS) in phosphate buffered saline (PBS). When
timing did not allow the B and T lymphocyte isolation to be completed immediately after the PBMC
separation, PBMCs were frozen in 1 ml freezing media (RPMI with 20% FBS and 10% dimethyl
sulfoxide (DMSO)) to be processed at a later time. The isolated CD3+ T lymphocytes were then frozen
at -80°C until DNA extraction.
Illumina Methylation 450K analysis
Based on the quality and quantity of the DNA isolated from T lymphocytes, 38 antepartum maternal
samples and 44 neonatal cord blood samples were selected for genome-wide DNA methylation analysis
with Infinium Human Methylation 450K BeadChip Array (WG-314-1001, Illumina Inc., San Diego,
CA, USA), which was performed at Genome Quebec, Montreal, Canada. For this analysis the genomic
DNA was quantified using Picogreen protocol (Quant-iT™ PicoGreen® dsDNA Products, P-7589,
Invitrogen, Carlsbad, CA, USA) and read on SpectraMAXGeminiXS Spectrophotometer. Bisulfite
conversion of 500 ng genomic DNA was carried out by the EZ-96 DNA Methylation-Gold Kit (D5007,
Zymo Research, Irvine, CA, USA).
The Illumina Methylation 450K kit was used for the microarray experiment following the
manufacturer’s protocol, with the exception that 8 µl of bisulfite converted material was utilized to
initiate the amplification step. An Illumina Hybridization oven was used for incubating amplified DNA
(37°C) and for BeadChips hybridization (48°C). A Hybex incubator was used for the fragmentation
(37°C) and the denaturation (95°C) steps. The X-stain step was carried out with a Tecan Freedom evo
robot with a Te-Flow module. Arrays were scanned on an Illumina iScan Reader.
All statistical analyses were carried out using the M-values as recommended by Du and colleagues51
because of the heteroscedasticity of the β-values. In the Illumina assay methylated signal corresponds to
Signal B and unmethylated signal corresponds to Signal A, therefore the β-value is calculated as: β =
max(Signal B,0) / max(Signal A,0)+max(Signal B,0)+100; whereas M-value is calculated as: M =
log2((max(Signal B,0)+1) / (max(Signal A,0)+1)).
Analyses of methylation data from hippocampal tissue samples
The brain tissue samples were collected from suicide completers and individuals who died suddenly in
an accident or by cardiac arrest and stored at the Quebec Suicide Brain Bank, Douglas Mental Health
Institute, Verdun, QC, Canada. Genomic DNA was extracted from the carefully dissected dentate gyrus
of the left hemisphere, and used for methylated DNA immunoprecipitation. After labeling the input and
the methylated DNA fractions to cyanine 3-dUTP (Cy3) and cyanine 5-dUTP (Cy5) using the Agilent
Genomic Enzymatic Labeling Kit, the samples were hybridized to a custom-designed 400K promoter
tiling array (Agilent Technologies, Santa Clara, CA, USA).33 Methylation levels of promoters were
obtained by taking the median estimated methylation level across each promoter. Differential
methylation between groups of samples was determined at the probe and promoter levels to ensure both
statistical significance and biological relevance, as previously described.52 At the probe level, a modified
t-statistic was computed for each probe corresponding to probe log-ratio differences using the ‘limma’
package of Bioconductor.53,54 Then, promoter-level methylation differences were calculated as
enrichment of large positive or negative t-statistic values among the probes in the promoter (about 10)
using the Wilcoxon rank-sum test.
Pyrosequencing
Genomic DNA of the neonatal cord blood T lymphocyte samples was quantified using Qubit dsDNA
BR Assay Kit (Q32853, Invitrogen, Carlsbad, CA, USA) and read on Qubit 2.0 Fluorometer (Q32866,
Invitrogen, Carlsbad, CA, USA). From 500 ng of genomic DNA the bisulfite conversion was done by
EZ DNA Methylation-Gold Kit (D5006, Zymo Research, Irvine, CA, USA). For one PCR amplification
~ 2% of the bisulfite converted DNA (equaling to ~ 10 ng of original genomic DNA) was used in 40 µl
reaction volume with 1 U of the EpiMark Hot Start Taq DNA Polymerase (M0490, New England
Biolabs, Ipswich, MA, USA) in the provided reaction buffer (final conc. 1x), 0.2 mM dNTPs and 0.2
µM primers. The thermocycle consisted of an initial denaturation step (95°C – 1 min), 45 cycles of
denaturation (95°C – 30 sec), annealing (58°C – 1 min), and extension (68°C – 1 min), and a final
extension step (68°C – 10 min). For the pyrosequencing 16 µl of the PCR products were used on either
PyroMark Q24 or Q96 (Qiagen, Venlo, Limburg, Netherlands) according to the manufacturer’s protocol.
The methylation percentage at each CpG site was analyzed using the PyroMark Q24 or CpG 1.0.11
software (Qiagen, Venlo, Limburg, Netherlands), then the level of methylation (β-value) was used to
calculate M-value as: M = log2(β/(1-β)). The sequences of primers used for pyrosequencing:
gene
BLK
CG-site
cg15742700
chr
8
position
11350853
FCER2
cg21121609,
cg12387247
cg19913465,
cg20234640
19
19
7766960,
7766974
7767075,
7767089
cg12002047
19
7767279
cg02606840
2
99280930
cg03429643
2
99280963
cg23217386
11
5653254
cg17976229
11
5653336
cg22160073
11
5653405
MGAT4A
TRIM34
forward primer
biot. reverse primer
sequencing primer
GATTGGATTGAGATAA
TATTGAGTTATGAG
GTTTGGAGTTTGTGTTT
GTTTTTTTAGTG
ATTTAAAACAATTAA
CCACATTCACATC
ACATCTCTAATTCTCA
CCCAATTCT
GTTGTAGGTTTGAT
AGGT
GTTTGTTTTTTTAGT
GTGTTG
GTTAAGTAGTAAGTTTT
TATAGGGTTGAAG
ACATTTAACCTAAAA
ACCTATTTACTCA
GTGGATGGGTGTGGTT
GATA
CCTCTAAAACAAACA
CCAAATAAACA
GAAGGGATATGTAAGG
AGAAAGTT
CTCTCTTCTAATTAAA
AACCCCTAAATA
TTTTATAGATTTAA
ATATGGGTTTG
TGTTATTGTATATT
GAATTATATTA
TGAGATAGTTGGTA
GGT
TGTATTTTGTATGT
AGTATTTTTAT
AGGAGAAAGTTGA
AAAAAG
AGTGTAAAAGTTTT
GAGGTA
TTTTTTTGTTTTTTG
ATATTGTAG
References
51. Du P, Zhang X, Huang CC, Jafari N, Kibbe WA, et al. (2010) Comparison of Beta-value and Mvalue methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics
11: 587.
52. Borghol N, Suderman M, McArdle W, Racine A, Hallett M, et al. (2012) Associations with earlylife socio-economic position in adult DNA methylation. Int J Epidemiol 41: 62-74.
53. Smyth GK (2005) Limma: linear models for microarray data. In: R. Gentleman VC, S. Dudoit, R.
Irizarry, W. Huber editor. Bioinformatics and Computational Biology Solutions using R and
Bioconductor. New York: Springer,. pp. 397-420.
54. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004) Bioconductor: open
software development for computational biology and bioinformatics. Genome Biol 5: R80.